This Project
This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.This project investigates the presence of the TANK gene in the whale shark genome, as well as establishes phylogeny of this gene in comparison to several species similar to humans and whale sharks.
Background Information
TRAF family member-associated NF-kappa-B activator is a protein in humans encoded by the TANK gene. The TRAF (tumor necrosis factor receptor-associated factor) family of proteins is part of a transduction mechanism of the tumor necrosis factor receptor superfamily (Pomerantz 1999). The protein is found in the cytoplasm and can bind to TRAF1, 2, or 3, silencing TRAF function by putting the proteins in a latent state in the cytoplasm. TRAF plays a role in the activation of the NF-KB signal transduction pathway (Figure 1). Three variants encoding different isoforms have been found for this gene, titled as TANK-X1, X2, and X3. (Roethe 1996) (Kaye 1996) (Chariot 2002).
Figure 1. A model for the activation of the NF-KB signal transduction pathway by the Tumor Necrosis Factor TNF-R2-TRAF signaling complex. This model shows how the I-TRAF mechanism functions as a general regulator of TRAF-mediated signaling events and the biological mechanism that results in NF-kappa B activation (Roethe 1996). NF-κB, involved with DNA transcription, is found in almost all animal cells, and is involved in cellular responses to stimuli such as free radicals, cytokines, UV light radiation, and bacterial or viral antigens (Gilmore 2006).
We were unable to identify a strong potential TANK ortholog from the newly-sequenced whale shark genome, but did identify that this gene was “lost” between the whale shark and it’s closely-related counterpart the elephant shark.
Methods Whale shark predicted orthologs
The human protein sequence (ENSP00000376505) was used as query in a Blast against the predicted whale shark protein database using the Galaxy server. Top predicted protein hits were then used as queries (using the full predicted sequence not only the aligned sequence) in protein BLASTs against the NCBI human protein database. The human protein sequence was also used in another Blast of the elephant shark genome.
Predicted orthologs
TANK predicted orthologs were identified in species other than whale sharks using the NCBI Blast server. Protein BLASTs were performed using single species protein databases for mouse, zebra fish, clawed frogs, chimpanzee, chicken, and elephant shark protein databases. The human TANK protein (ENSP00000376505) was used as query sequence in these searches with default settings.
Phylogenetic tree
The hit with the lowest E-value for each non-whale shark species search (using the human protein as query) along with the top 4 whale shark BLAST hits were used to create a multiple sequence alignment and phylogenetic tree. ClustalW2 with default settings was used to create the alignment and tree.
Tank Investigation in Whale Shark Genome
Searching for TANK protein sequence was used to query the whale shark predicted protein database and results are shown in Table 1. There were 5 hits with e-values less than .001. These 5 best hits were then Blasted against the human protein database using NCBI BLASTp.
Sequence ID
Alignment Length
% Identity Coverage
E-value
Query Length
g26587.t1
78
18.35
5e-5
425
g15269.t1
60
14.12
5e-5
425
g30944.t1
71
16.71
7e-5
425
g45125.t1
52
12.24
5e-5
425
g40674.t1
90
21.16
5e-4
425
Table 1. Human TANK BLASTp best hits against the whale shark predicted protein database. The Galaxy server was used to query the predicted whale shark protein database using the human TANK protein sequence. The top 5 hits according to E-value are reported here with their database sequence ID, amino acid length, and percentage identity coverage. These sequences were also used as queries against the NCBI human protein database.
Since none of the predicted proteins from the Blast of the whale shark proteins using the human TANK sequence as query returned TANK as the best hit against the human protein database, we were not confident that any of these five were TANK orthologs (Table 1). When comparing the human TANK against the elephant shark, there were several suitable matches for a predicted TANK. We then repeated this process using the Elephant Shark predicted TANK protein as query against the whale shark predicted protein database to see if a more closely related species would return different best hits. There were no hits that returned TANK as the best hit again.
Protein domains
Potential TANK-like proteins in the whale shark (from BLAST results) were Blasted with the human genome.Three of the five top potential TANK-likes were contained within the RT-nLTR-like superfamily (Figure 2). The retrovirus reverse transcriptase (RT) non-LTR (long terminal repeat) retrotransposon superfamily catalyzes the conversion of single-stranded RNA into double-stranded DNA for integration into host chromosomes (Marchler-Bauer et al 2015). This protein domain did not match the human TANK's protein domain, which was the TBD superfamily.
The Tbk1/Ikki binding domain (TBD) is a 40 amino acid protein domain that is able to bind kinases together, and is a primary identifier in the TANK gene. This superfamily is predicted to form an alpha-helix with kinase-binding factors clustering to one side.
Figure 2.Putative domains of whale shark potential TANK-like best hit predicted proteins.Three of the five top sequences from the whale shark genome were in the RT-nLTR-like superfamily. Two of these three returned a predicted TANK identity when re-Blasted against the human genome.
Orthologs
The human TANK protein sequence (ENSP00000376505) was used as query in NCBI BLAST searches against individual species' protein databases. TANK matches were found using this method in all tested animals. For the whale shark, the closest potential-TANK sequence was Blasted with human to see if it returned as TANK, which it did not. Furthermore, comparing these orthologs to the whale shark did not reveal any matches listed as TANK that had E-values less than 0.001.
Species
Name
Length
% Identity Coverage
E-value
Sequence ID
Elephant shark
TRAF family member-associated NF-kappa-B activator
86
28
9e-23
XP_007888043.1
Zebrafish
TRAF family member-associated NF-kappa-B activator
76
30
6e-19
NP_001070068.1
Mouse
TRAF family member-associated NF-kappa-B activator isoform X3
236
82
8e-172
XP_006499152.1
Clawed Frog
TRAF family member-associated NF-kappa-B activator isoform X2
104
35
1e-40
XP_004917754.1
Chicken
TRAF family member-associated NF-kappa-B activator
154
49
4e-88
NP_001264800.1
Chimpanzee
TRAF family member-associated NF-kappa-B activator isoform X2
422
99
0
XP_001150111.2
Whale Shark
matrilin-4 isoform 3 precursor
115
63
8e-84
NP_085095.1
Table 2. Best hits with human TANK protein BLAST. The human TANK sequence was used in protein BLASTs against individual species. Name, ID, length, percent identity coverage, and E-value of the best hit from each search are reported here. Whale shark was the only animal that did not return TANK, in any isoform or subclass.
Phylogeny
The top hits from the protein database using the human TANK protein as query were used to create a phylogenetic tree. From this tree, it is evident that the TANK protein is highly prevalent in nearly all of the species except the whale shark (Figure 2). The fact that the divergence occurs only with the whale shark is fascinating. This possibly suggests that whale sharks may have adapted over time to not need the TANK gene. The reason for this adaptation or mutation is unknown, but further investigation into this matter may reveal interesting results.
Figure 3. Phylogenetic tree of human TANK protein among various animals. The best hit from BLAST searches of protein databases were used in the ClustalW2 program to create a phylogenetic tree. For the whale shark, the two best hits from the top five that were in the RT-like superfamily were chosen (Figure 2). The two ID numbers at the bottom are the two whale shark potential-TANK sequences. Branch lengths represent relative evolutionary time.
Conclusions
While we were not able to identify a predicted TANK ortholog in whale sharks, we were able to find evidence for where the TANK gene diverged. According to the phylogenetic tree and the ortholog comparisons, the TANK gene diverged at the common ancestor of the whale shark and elephant shark. The predicted TANK ortholog in the elephant shark does suggest that an TANK ortholog exists in sharks, but somewhere diverged for the whale shark. More research must be done to explore the absence of the TANK gene in the whale shark and any other genes that have similar functions that do exist in this creature.
References
Rothe M, Xiong J, Shu HB, Williamson K, Goddard A, Goeddel DV (September 1996). "I-TRAF is a novel TRAF-interacting protein that regulates TRAF-mediated signal transduction". Proc Natl Acad Sci U S A 93 (16): 8241–6.
Kaye KM, Devergne O, Harada JN, Izumi KM, Yalamanchili R, Kieff E, Mosialos G (November 1996). "Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein". Proc Natl Acad Sci U S A 93 (20): 11085–90.
Chariot, Alain; Leonardi Antonio; Muller Jurgen; Bonif Marianne; Brown Keith; Siebenlist Ulrich (October 2002). "Association of the adaptor TANK with the I kappa B kinase (IKK) regulator NEMO connects IKK complexes with IKK epsilon and TBK1 kinases". J. Biol. Chem. (United States) 277 (40): 37029–36.
Pomerantz, J L; Baltimore D (December 1999). "NF-kappaB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase". EMBO J. (ENGLAND) 18 (23): 6694–704.
Bouwmeester, Tewis; Bauch Angela, Ruffner Heinz, Angrand Pierre-Olivier, Bergamini Giovanna, Croughton Karen, Cruciat Cristina, Eberhard Dirk, Gagneur Julien, Ghidelli Sonja, Hopf Carsten, Huhse Bettina, Mangano Raffaella, Michon Anne-Marie, Schirle Markus, Schlegl Judith, Schwab Markus, Stein Martin A, Bauer Andreas, Casari Georg, Drewes Gerard, Gavin Anne-Claude, Jackson David B, Joberty Gerard, Neubauer Gitte, Rick Jens, Kuster Bernhard, Superti-Furga Giulio (February 2004). "A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway". Nat. Cell Biol. (England) 6 (2): 97–105.
This Project
This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.This project investigates the presence of the TANK gene in the whale shark genome, as well as establishes phylogeny of this gene in comparison to several species similar to humans and whale sharks.
Background Information
TRAF family member-associated NF-kappa-B activator is a protein in humans encoded by the TANK gene. The TRAF (tumor necrosis factor receptor-associated factor) family of proteins is part of a transduction mechanism of the tumor necrosis factor receptor superfamily (Pomerantz 1999). The protein is found in the cytoplasm and can bind to TRAF1, 2, or 3, silencing TRAF function by putting the proteins in a latent state in the cytoplasm. TRAF plays a role in the activation of the NF-KB signal transduction pathway (Figure 1). Three variants encoding different isoforms have been found for this gene, titled as TANK-X1, X2, and X3. (Roethe 1996) (Kaye 1996) (Chariot 2002).
Figure 1. A model for the activation of the NF-KB signal transduction pathway by the Tumor Necrosis Factor TNF-R2-TRAF signaling complex. This model shows how the I-TRAF mechanism functions as a general regulator of TRAF-mediated signaling events and the biological mechanism that results in NF-kappa B activation (Roethe 1996). NF-κB, involved with DNA transcription, is found in almost all animal cells, and is involved in cellular responses to stimuli such as free radicals, cytokines, UV light radiation, and bacterial or viral antigens (Gilmore 2006).
We were unable to identify a strong potential TANK ortholog from the newly-sequenced whale shark genome, but did identify that this gene was “lost” between the whale shark and it’s closely-related counterpart the elephant shark.
Methods
Whale shark predicted orthologs
The human protein sequence (ENSP00000376505) was used as query in a Blast against the predicted whale shark protein database using the Galaxy server. Top predicted protein hits were then used as queries (using the full predicted sequence not only the aligned sequence) in protein BLASTs against the NCBI human protein database. The human protein sequence was also used in another Blast of the elephant shark genome.
Predicted orthologs
TANK predicted orthologs were identified in species other than whale sharks using the NCBI Blast server. Protein BLASTs were performed using single species protein databases for mouse, zebra fish, clawed frogs, chimpanzee, chicken, and elephant shark protein databases. The human TANK protein (ENSP00000376505) was used as query sequence in these searches with default settings.
Phylogenetic tree
The hit with the lowest E-value for each non-whale shark species search (using the human protein as query) along with the top 4 whale shark BLAST hits were used to create a multiple sequence alignment and phylogenetic tree. ClustalW2 with default settings was used to create the alignment and tree.
Tank Investigation in Whale Shark Genome
Searching for TANK protein sequence was used to query the whale shark predicted protein database and results are shown in Table 1. There were 5 hits with e-values less than .001. These 5 best hits were then Blasted against the human protein database using NCBI BLASTp.
Since none of the predicted proteins from the Blast of the whale shark proteins using the human TANK sequence as query returned TANK as the best hit against the human protein database, we were not confident that any of these five were TANK orthologs (Table 1). When comparing the human TANK against the elephant shark, there were several suitable matches for a predicted TANK. We then repeated this process using the Elephant Shark predicted TANK protein as query against the whale shark predicted protein database to see if a more closely related species would return different best hits. There were no hits that returned TANK as the best hit again.
Protein domains
Potential TANK-like proteins in the whale shark (from BLAST results) were Blasted with the human genome.Three of the five top potential TANK-likes were contained within the RT-nLTR-like superfamily (Figure 2). The retrovirus reverse transcriptase (RT) non-LTR (long terminal repeat) retrotransposon superfamily catalyzes the conversion of single-stranded RNA into double-stranded DNA for integration into host chromosomes (Marchler-Bauer et al 2015). This protein domain did not match the human TANK's protein domain, which was the TBD superfamily.
The Tbk1/Ikki binding domain (TBD) is a 40 amino acid protein domain that is able to bind kinases together, and is a primary identifier in the TANK gene. This superfamily is predicted to form an alpha-helix with kinase-binding factors clustering to one side.
Figure 2. Putative domains of whale shark potential TANK-like best hit predicted proteins.Three of the five top sequences from the whale shark genome were in the RT-nLTR-like superfamily. Two of these three returned a predicted TANK identity when re-Blasted against the human genome.
Orthologs
The human TANK protein sequence (ENSP00000376505) was used as query in NCBI BLAST searches against individual species' protein databases. TANK matches were found using this method in all tested animals. For the whale shark, the closest potential-TANK sequence was Blasted with human to see if it returned as TANK, which it did not. Furthermore, comparing these orthologs to the whale shark did not reveal any matches listed as TANK that had E-values less than 0.001.
Phylogeny
The top hits from the protein database using the human TANK protein as query were used to create a phylogenetic tree. From this tree, it is evident that the TANK protein is highly prevalent in nearly all of the species except the whale shark (Figure 2). The fact that the divergence occurs only with the whale shark is fascinating. This possibly suggests that whale sharks may have adapted over time to not need the TANK gene. The reason for this adaptation or mutation is unknown, but further investigation into this matter may reveal interesting results.
Figure 3. Phylogenetic tree of human TANK protein among various animals. The best hit from BLAST searches of protein databases were used in the ClustalW2 program to create a phylogenetic tree. For the whale shark, the two best hits from the top five that were in the RT-like superfamily were chosen (Figure 2). The two ID numbers at the bottom are the two whale shark potential-TANK sequences. Branch lengths represent relative evolutionary time.
Conclusions
While we were not able to identify a predicted TANK ortholog in whale sharks, we were able to find evidence for where the TANK gene diverged. According to the phylogenetic tree and the ortholog comparisons, the TANK gene diverged at the common ancestor of the whale shark and elephant shark. The predicted TANK ortholog in the elephant shark does suggest that an TANK ortholog exists in sharks, but somewhere diverged for the whale shark. More research must be done to explore the absence of the TANK gene in the whale shark and any other genes that have similar functions that do exist in this creature.
Resources:
Ensembl Genome Sequence Extractor: http://www.ensembl.org/index.html
NCBI BLAST Website: http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome
Whale Shark Galaxy Server: http://whaleshark.georgiaaquarium.org/
CLUSTALW Phylogenetic Tree: http://www.genome.jp/tools/clustalw/
References
Rothe M, Xiong J, Shu HB, Williamson K, Goddard A, Goeddel DV (September 1996). "I-TRAF is a novel TRAF-interacting protein that regulates TRAF-mediated signal transduction". Proc Natl Acad Sci U S A 93 (16): 8241–6.
Kaye KM, Devergne O, Harada JN, Izumi KM, Yalamanchili R, Kieff E, Mosialos G (November 1996). "Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein". Proc Natl Acad Sci U S A 93 (20): 11085–90.
Chariot, Alain; Leonardi Antonio; Muller Jurgen; Bonif Marianne; Brown Keith; Siebenlist Ulrich (October 2002). "Association of the adaptor TANK with the I kappa B kinase (IKK) regulator NEMO connects IKK complexes with IKK epsilon and TBK1 kinases". J. Biol. Chem. (United States) 277 (40): 37029–36.
Pomerantz, J L; Baltimore D (December 1999). "NF-kappaB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase". EMBO J. (ENGLAND) 18 (23): 6694–704.
Bouwmeester, Tewis; Bauch Angela, Ruffner Heinz, Angrand Pierre-Olivier, Bergamini Giovanna, Croughton Karen, Cruciat Cristina, Eberhard Dirk, Gagneur Julien, Ghidelli Sonja, Hopf Carsten, Huhse Bettina, Mangano Raffaella, Michon Anne-Marie, Schirle Markus, Schlegl Judith, Schwab Markus, Stein Martin A, Bauer Andreas, Casari Georg, Drewes Gerard, Gavin Anne-Claude, Jackson David B, Joberty Gerard, Neubauer Gitte, Rick Jens, Kuster Bernhard, Superti-Furga Giulio (February 2004). "A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway". Nat. Cell Biol. (England) 6 (2): 97–105.
Nomura, F; Kawai T; Nakanishi K; Akira S (March 2000). "NF-kappaB activation through IKK-i-dependent I-TRAF/TANK phosphorylation". Genes Cells (ENGLAND) 5 (3): 191–202.
Gilmore TD (2006). "Introduction to NF-κB: players, pathways, perspectives". Oncogene 25 (51): 6680–4.
Marchler-Bauer A et al. (2015), "CDD: NCBI's conserved domain database.", Nucleic Acids Res.43(D)222-6.